The importance of 3D fibre architecture in cancer and implications for biomaterial model design.

The need for improved prediction of clinical response is driving the development of cancer models with enhanced physiological relevance. A new concept of 'precision biomaterials' is emerging, encompassing patient-mimetic biomaterial models that seek to accurately detect, treat and model cancer by faithfully recapitulating key microenvironmental characteristics. Despite recent advances allowing tissue-mimetic stiffness and molecular composition to be replicated in vitro, approaches for reproducing the 3D fibre architectures found in tumour extracellular matrix (ECM) remain relatively unexplored. Although the precise influences of patient-specific fibre architecture are unclear, we summarize the known roles of tumour fibre architecture, underlining their implications in cell-matrix interactions and ultimately clinical outcome. We then explore the challenges in reproducing tissue-specific 3D fibre architecture(s) in vitro, highlighting relevant biomaterial fabrication techniques and their benefits and limitations. Finally, we discuss imaging and image analysis techniques (focussing on collagen I-optimized approaches) that could hold the key to mapping tumour-specific ECM into high-fidelity biomaterial models. We anticipate that an interdisciplinary approach, combining materials science, cancer research and image analysis, will elucidate the role of 3D fibre architecture in tumour development, leading to the next generation of patient-mimetic models for mechanistic studies and drug discovery.

Silk fibroin increases the elasticity of alginate-gelatin hydrogels and regulates cardiac cell contractile function in cardiac bioinks.

Silk fibroin (SF) is a natural protein extracted fromBombyx morisilkworm thread. From its common use in the textile industry, it emerged as a biomaterial with promising biochemical and mechanical properties for applications in the field of tissue engineering and regenerative medicine. In this study, we evaluate for the first time the effects of SF on cardiac bioink formulations containing cardiac spheroids (CSs). First, we evaluate if the SF addition plays a role in the structural and elastic properties of hydrogels containing alginate (Alg) and gelatin (Gel). Then, we test the printability and durability of bioprinted SF-containing hydrogels. Finally, we evaluate whether the addition of SF controls cell viability and function of CSs in Alg-Gel hydrogels. Our findings show that the addition of 1% (w/v) SF to Alg-Gel hydrogels makes them more elastic without affecting cell viability. However, fractional shortening (FS%) of CSs in SF-Alg-Gel hydrogels increases without affecting their contraction frequency, suggesting an improvement in contractile function in the 3D cultures. Altogether, our findings support a promising pathway to bioengineer bioinks containing SF for cardiac applications, with the ability to control mechanical and cellular features in cardiac bioinks.

Correction: Spatial variation and antecedent sea surface temperature conditions influence Hawaiian intertidal community structure.

[This corrects the article DOI: 10.1371/journal.pone.0286136.].

Spatial variation and antecedent sea surface temperature conditions influence Hawaiian intertidal community structure.

Global sea surface temperatures (SSTs) are increasing, and in Hawai'i, rates of ocean warming are projected to double by the end of the 21st century. However, current nearshore warming trends and their possible impacts on intertidal communities are not well understood. This study represents the first investigation into the possible effects of rising SST on intertidal algal and invertebrate communities across the Main Hawaiian Islands (MHI). By utilizing citizen-science data coupled with high-resolution, daily SST satellite measurements from 12 intertidal sites across the MHI from 2004-2019, the response of intertidal algal and invertebrate abundance and community diversity to changes in SST was investigated across multiple spatial scales. Results show high rates of SST warming (0.40°C Decade-1) over this study's timeframe, similar to predicted rates of warming for Hawai'i by the end of the 21st century. Changes in abundance and diversity in response to SST were variable among intertidal sites, but differences in antecedent SST among intertidal sites were significantly associated with community dissimilarity. In addition, a statistically significant positive relationship was found between SST and Simpson's diversity index, and a significant relationship was also found between SST and the abundance of six dominant taxa. For five of these six dominant taxa, antecedent SSTs over the 6-12 months preceding sampling were the most influential for describing changes to abundance. The increase in community diversity in response to higher SSTs was best explained by temperatures in the 10 months preceding sampling, and the resultant decreased abundance of dominant turf algae. These results highlight rapidly warming nearshore SSTs in Hawai'i and the longer-term effects of antecedent SSTs as significant drivers of change within Hawaiian intertidal communities. Therefore, we suggest that future research and management should consider the possibility of lagging effects of antecedent SST on intertidal communities in Hawai'i and elsewhere.

Patient centered guidelines for the laboratory diagnosis of Gaucher disease type 1.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder due to the deficient activity of the acid beta-glucosidase (GCase) enzyme, resulting in the progressive lysosomal accumulation of glucosylceramide (GlcCer) and its deacylated derivate, glucosylsphingosine (GlcSph). GCase is encoded by the GBA1 gene, located on chromosome 1q21 16 kb upstream from a highly homologous pseudogene. To date, more than 400 GBA1 pathogenic variants have been reported, many of them derived from recombination events between the gene and the pseudogene. In the last years, the increased access to new technologies has led to an exponential growth in the number of diagnostic laboratories offering GD testing. However, both biochemical and genetic diagnosis of GD are challenging and to date no specific evidence-based guidelines for the laboratory diagnosis of GD have been published. The objective of the guidelines presented here is to provide evidence-based recommendations for the technical implementation and interpretation of biochemical and genetic testing for the diagnosis of GD to ensure a timely and accurate diagnosis for patients with GD worldwide. The guidelines have been developed by members of the Diagnostic Working group of the International Working Group of Gaucher Disease (IWGGD), a non-profit network established to promote clinical and basic research into GD for the ultimate purpose of improving the lives of patients with this disease. One of the goals of the IWGGD is to support equitable access to diagnosis of GD and to standardize procedures to ensure an accurate diagnosis. Therefore, a guideline development group consisting of biochemists and geneticists working in the field of GD diagnosis was established and a list of topics to be discussed was selected. In these guidelines, twenty recommendations are provided based on information gathered through a systematic review of the literature and two different diagnostic algorithms are presented, considering the geographical differences in the access to diagnostic services. Besides, several gaps in the current diagnostic workflow were identified and actions to fulfill them were taken within the IWGGD. We believe that the implementation of recommendations provided in these guidelines will promote an equitable, timely and accurate diagnosis for patients with GD worldwide.

Immune signatures underlying post-acute COVID-19 lung sequelae.

Severe coronavirus disease 2019 (COVID-19) pneumonia survivors often exhibit long-term pulmonary sequelae, but the underlying mechanisms or associated local and systemic immune correlates are not known. Here, we have performed high-dimensional characterization of the pathophysiological and immune traits of aged COVID-19 convalescents, and correlated the local and systemic immune profiles with pulmonary function and lung imaging. We found that chronic lung impairment was accompanied by persistent respiratory immune alterations. We showed that functional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)­specific memory T and B cells were enriched at the site of infection compared with those of blood. Detailed evaluation of the lung immune compartment revealed that dysregulated respiratory CD8+ T cell responses were associated with the impaired lung function after acute COVID-19. Single-cell transcriptomic analysis identified the potential pathogenic subsets of respiratory CD8+ T cells contributing to persistent tissue conditions after COVID-19. Our results have revealed pathophysiological and immune traits that may support the development of lung sequelae after SARS-CoV-2 pneumonia in older individuals, with implications for the treatment of chronic COVID-19 symptoms.

Imaging of lymphatic dysplasia in Noonan syndrome: Case studies and historical atlas.

To determine the historical use and utility of various lymphatic imaging modalities in Noonan syndrome (NS) patients, we performed a comprehensive literature review by collecting the published medical imaging of NS lymphatic dysplasias. We correlated imaging findings with clinical phenotypes and treatment. Our analysis of lymphatic imaging modalities provides an algorithmic approach to imaging and patient care across the spectrum of NS developmental defects. A total of 54 NS cases have been published since 1975. Using the observations reported in 15 reviewed publications, an association was made between disruptions in central lymphatic flow and poor clinical presentations/outcomes in NS patients.

Comparative evolution of vegetative branching in sorghum.

Tillering and secondary branching are two plastic traits with high agronomic importance, especially in terms of the ability of plants to adapt to changing environments. We describe a quantitative trait analysis of tillering and secondary branching in two novel BC1F2 populations totaling 246 genotypes derived from backcrossing two Sorghum bicolor x S. halepense F1 plants to a tetraploidized S. bicolor. A two-year, two-environment phenotypic evaluation in Bogart, GA and Salina, KS permitted us to identify major effect and environment specific QTLs. Significant correlation between tillering and secondary branching followed by discovery of overlapping sets of QTLs continue to support the developmental relationship between these two organs and suggest the possibility of pleiotropy. Comparisons with two other populations sharing S. bicolor BTx623 as a common parent but sampling the breadth of the Sorghum genus, increase confidence in QTL detected for these two plastic traits and provide insight into the evolution of morphological diversity in the Eusorghum clade. Correspondence between flowering time and vegetative branching supports other evidence in suggesting a pleiotropic effect of flowering genes. We propose a model to predict biomass weight from plant architecture related traits, quantifying contribution of each trait to biomass and providing guidance for future breeding experiments.

Automated annotation and visualisation of high-resolution spatial proteomic mass spectrometry imaging data using HIT-MAP.

Spatial proteomics has the potential to significantly advance our understanding of biology, physiology and medicine. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool in the spatial proteomics field, enabling direct detection and registration of protein abundance and distribution across tissues. MALDI-MSI preserves spatial distribution and histology allowing unbiased analysis of complex, heterogeneous tissues. However, MALDI-MSI faces the challenge of simultaneous peptide quantification and identification. To overcome this, we develop and validate HIT-MAP (High-resolution Informatics Toolbox in MALDI-MSI Proteomics), an open-source bioinformatics workflow using peptide mass fingerprint analysis and a dual scoring system to computationally assign peptide and protein annotations to high mass resolution MSI datasets and generate customisable spatial distribution maps. HIT-MAP will be a valuable resource for the spatial proteomics community for analysing newly generated and retrospective datasets, enabling robust peptide and protein annotation and visualisation in a wide array of normal and disease contexts.

The rare C9 P167S risk variant for age-related macular degeneration increases polymerization of the terminal component of the complement cascade.

Age-related macular degeneration (AMD) is a complex neurodegenerative eye disease with behavioral and genetic etiology and is the leading cause of irreversible vision loss among elderly Caucasians. Functionally significant genetic variants in the alternative pathway of complement have been strongly linked to disease. More recently, a rare variant in the terminal pathway of complement has been associated with increased risk, Complement component 9 (C9) P167S. To assess the functional consequence of this variant, C9 levels were measured in two independent cohorts of AMD patients. In both cohorts, it was demonstrated that the P167S variant was associated with low C9 plasma levels. Further analysis showed that patients with advanced AMD had elevated sC5b-9 compared to those with non-advanced AMD, although this was not associated with the P167S polymorphism. Electron microscopy of membrane attack complexes (MACs) generated using recombinantly produced wild type or P167S C9 demonstrated identical MAC ring structures. In functional assays, the P167S variant displayed a higher propensity to polymerize and a small increase in its ability to induce hemolysis of sheep erythrocytes when added to C9-depleted serum. The demonstration that this C9 P167S AMD risk polymorphism displays increased polymerization and functional activity provides a rationale for the gene therapy trials of sCD59 to inhibit the terminal pathway of complement in AMD that are underway.

Quantitative trait mapping of plant architecture in two BC1F2 populations of Sorghum Bicolor × S. halepense and comparisons to two other sorghum populations.

KEY MESSAGE: Comparing populations derived, respectively, from polyploid Sorghum halepense and its progenitors improved knowledge of plant architecture and showed that S. halepense harbors genetic novelty of potential value for sorghum improvement Vegetative growth and the timing of the vegetative-to-reproductive transition are critical to a plant's fitness, directly and indirectly determining when and how a plant lives, grows and reproduces. We describe quantitative trait analysis of plant height and flowering time in the naturally occurring tetraploid Sorghum halepense, using two novel BC1F2 populations totaling 246 genotypes derived from backcrossing two tetraploid Sorghum bicolor x S. halepense F1 plants to a tetraploidized S. bicolor. Phenotyping for two years each in Bogart, GA and Salina, KS allowed us to dissect variance into narrow-sense genetic (QTLs) and environmental components. In crosses with a common S. bicolor BTx623 parent, comparison of QTLs in S. halepense, its rhizomatous progenitor S. propinquum and S. bicolor race guinea which is highly divergent from BTx623 permit inferences of loci at which new alleles have been associated with improvement of elite sorghums. The relative abundance of QTLs unique to the S. halepense populations may reflect its polyploidy and subsequent 'diploidization' processes often associated with the formation of genetic novelty, a possibility further supported by a high level of QTL polymorphism within sibling lines derived from a common S. halepense parent. An intriguing hypothesis for further investigation is that polyploidy of S. halepense following 96 million years of abstinence, coupled with natural selection during its spread to diverse environments across six continents, may provide a rich collection of novel alleles that offer potential opportunities for sorghum improvement.

Investigation of fibrillin microfibrils in the canine cruciate ligament in dogs with different predispositions to ligament rupture.

Cranial cruciate ligament disease (CCLD) is the most common cause of pelvic limb lameness in dogs but its precise aetiopathogenesis is uncertain. Fibrillin microfibrils (FM) are complex macro-molecular assemblies found in many tissues including ligaments, where they are thought to play an important mechanical role. We hypothesised that FM ultrastructural variation correlates with the differing predisposition of canine breeds to CCLD. Non-diseased cranial and caudal cruciate ligaments (CCLs and CaCLs) were obtained from Greyhound (GH) and Staffordshire Bull Terrier (SBT) cadavers. Fibrillin microfibrils were extracted from the ligaments by bacterial collagenase digestion, purified by size-exclusion chromatography and subsequently visualized by atomic force microscopy (AFM). With AFM, FMs have a characteristic beads-on-a-string appearance. For each FM, periodicity (bead-bead distance) and length (number of beads/FM) was measured. Fibrillin microfibril length was found to be similar for GH and SBT, with non-significant inter-breed and inter-ligament differences. Fibrillin microfibril periodicity varied when comparing GH and SBT for CCL (GH 60.2 ± 1.4 nm; SBT 56.2 ± 0.8 nm) and CaCL (GH 55.5 ± 1.6 nm; SBT 61.2 ± 1.2 nm). A significant difference was found in the periodicity distribution when comparing CCL for both breeds (P < 0.00001), further, intra-breed differences in CCL vs CaCL were statistically significant within both breeds (P < 0.00001). The breed at low risk of CCLD exhibited a periodicity profile which may be suggestive of a repair and remodelling within the CCL.

The Evolution of an Invasive Plant, Sorghum halepense L. ('Johnsongrass').

From noble beginnings as a prospective forage, polyploid Sorghum halepense ('Johnsongrass') is both an invasive species and one of the world's worst agricultural weeds. Formed by S. bicolor x S. propinquum hybridization, we show S. halepense to have S. bicolor-enriched allele composition and striking mutations in 5,957 genes that differentiate it from representatives of its progenitor species and an outgroup. The spread of S. halepense may have been facilitated by introgression from closely-related cultivated sorghum near genetic loci affecting rhizome development, seed size, and levels of lutein, a photochemical protectant and abscisic acid precursor. Rhizomes, subterranean stems that store carbohydrates and spawn clonal propagules, have growth correlated with reproductive rather than other vegetative tissues, and increase survival of both temperate cold seasons and tropical dry seasons. Rhizomes of S. halepense are more extensive than those of its rhizomatous progenitor S. propinquum, with gene expression including many alleles from its non-rhizomatous S. bicolor progenitor. The first surviving polyploid in its lineage in ∼96 million years, its post-Columbian spread across six continents carried rich genetic diversity that in the United States has facilitated transition from agricultural to non-agricultural niches. Projected to spread another 200-600 km northward in the coming century, despite its drawbacks S. halepense may offer novel alleles and traits of value to improvement of sorghum.

Transmission Genetics of a Sorghum bicolor × S. halepense Backcross Populations.

Despite a "ploidy barrier," interspecific crosses to wild and/or cultivated sorghum (Sorghum bicolor, 2n = 2x = 20) may have aided the spread across six continents of Sorghum halepense, also exemplifying risks of "transgene escape" from crops that could make weeds more difficult to control. Genetic maps of two BC1F1 populations derived from crosses of S. bicolor (sorghum) and S. halepense with totals of 722 and 795 single nucleotide polymorphism (SNP) markers span 37 and 35 linkage groups, with 2-6 for each of the 10 basic sorghum chromosomes due to fragments covering different chromosomal portions or independent segregation from different S. halepense homologs. Segregation distortion favored S. halepense alleles on chromosomes 2 (1.06-4.68 Mb, near a fertility restoration gene), 7 (1.20-6.16 Mb), 8 (1.81-5.33 Mb, associated with gene conversion), and 9 (47.5-50.1 Mb); and S. bicolor alleles on chromosome 6 (0-40 Mb), which contains both a large heterochromatin block and the Ma1 gene. Regions of the S. halepense genome that are recalcitrant to gene flow from sorghum might be exploited as part a multi-component system to reduce the likelihood of spread of transgenes or other modified genes. Its SNP profile suggests that chromosome segments from its respective progenitors S. bicolor and Sorghum propinquum have extensively recombined in S. halepense. This study reveals genomic regions that might discourage crop-to-weed gene escape, and provides a foundation for marker-trait association analysis to determine the genetic control of traits contributing to weediness, invasiveness, and perenniality of S. halepense.

Personal protection and delivery of rhinologic and endoscopic skull base procedures during the COVID-19 outbreak.

On March 11th 2020, the World Health Organization (WHO) declared COVID-19 pandemic, with subsequent profound impact on the entire health care system. During the COVID-19 outbreak, activities in the rhinology outpatient clinic and operation rooms are limited to emergency care only. Health care practitioners are faced with the need to perform rhinological and skull base emergency procedures in patients with a positive or unknown COVID-19 status. This article aims to provide recommendations and relevant information for rhinologists, based on the limited amount of (anecdotal) data, to guarantee high-quality patient care and adequate levels of infection prevention in the rhinology clinic.

Critical transitions in suspended sediment dynamics in a temperate meso-tidal estuary.

There is growing consensus that human interventions can fundamentally change fine sediment transport in estuaries. Critical transitions in response to human interventions have been hypothesized based on indirect observational evidence and theoretical understanding. So far direct evidence has been lacking. Based on a 20 year data-set of surface suspended particulate matter (SPM) concentrations, we present empirical evidence of critical transitions in a temperate meso-tidal estuary. In 2008-2009 the SPM dynamics of the Scheldt estuary (Belgium/The Netherlands) changed dramatically. Not only did the total amount of sediment in suspension increase, a new maximum turbidity zone (MTZ) at typical winter discharges appeared. At intermediate and low summer discharges the longitudinal distribution of SPM now flickers between two markedly different states. Our data suggest that a range of human interventions (fairway widening and deepening, dredging and dumping activities) set the scene leading to the observed transitions. Moreover the freshwater MTZ in the Scheldt and in its major tributary exhibit an increasing sensitivity towards freshwater discharge, coinciding with water quality improvements. This suggests large scale impacts of changes in eutrophication status on estuarine sediment dynamics. This has largely been a blind spot in morphodynamic research.

Quantification of the flux of tyrosine pathway metabolites during nitisinone treatment of Alkaptonuria.

Nitisinone decreases homogentisic acid (HGA) in Alkaptonuria (AKU) by inhibiting the tyrosine metabolic pathway in humans. The effect of different daily doses of nitisinone on circulating and 24 h urinary excretion of phenylalanine (PA), tyrosine (TYR), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and HGA in patients with AKU was studied over a four week period. Forty AKU patients, randomised into five groups of eight patients, received doses of 1, 2, 4 or 8 mg of nitisinone daily, or no drug (control). Metabolites were analysed by tandem mass spectrometry in 24 h urine and serum samples collected before and after nitisinone. Serum metabolites were corrected for total body water and the sum of 24 hr urine plus total body water metabolites of PA, TYR, HPPA, HPLA and HGA were determined. Body weight and urine urea were used to check on stability of diet and metabolism over the 4 weeks of study. The sum of quantities of urine metabolites (PA, TYR, HPPA, HPLA and HGA) were similar pre- and post-nitisinone. The sum of total body water metabolites were significantly higher post-nitisinone (p < 0.0001) at all doses. Similarly, combined 24 hr urine:total body water ratios for all analytes were significantly higher post-nitisinone, compared with pre-nitisinone baseline for all doses (p = 0.0002 - p < 0.0001). Significantly higher concentrations of metabolites from the tyrosine metabolic pathway were observed in a dose dependant manner following treatment with nitisinone and we speculate that, for the first time, experimental evidence of the metabolite pool that would otherwise be directed towards pigment formation, has been unmasked.

Aboriginal community understandings of dementia and responses to dementia care.

OBJECTIVE: Aboriginal people and Torres Strait Islanders are impacted by dementia at higher rates and at a younger age of onset than the broader Australia population. Public health strategies to support this population require a thorough understanding of how Aboriginal people perceive dementia and dementia care support needs. The aim of this study was to investigate Aboriginal community understandings of dementia and their responses to dementia care. STUDY DESIGN: This study is a community participatory action research partnership. METHODS: The mixed method study was undertaken in 2017 with members of a discrete Aboriginal community from rural Tasmania, Australia. Participants were older than 18 years, self-identified as an Aboriginal person and were living or had lived in the community studied. Data were derived from 50 participants who completed the 27-item Dementia Knowledge Assessment Scale (DKAS). Twelve of these participants also shared their dementia care experiences in individual interviews. RESULTS: The DKAS results showed a low overall level of dementia knowledge, with a total mean score of 27.73 (scoring 51% on average) of a possible score of 54. The salient interview theme was the cultural obligation to care for family members living with dementia. Dementia care metaphors represented ways to protect family members living with dementia and maintain their ongoing connections to home and community. However, limited understandings of dementia affected their capacity to provide quality care. CONCLUSION: The findings contribute to public health scholarship involving Aboriginal community responses to dementia care. The study has resulted in important initiatives including a community-based dementia education program to ensure Aboriginal people impacted by dementia are provided with effective and culturally appropriate care.

Ocean acidification affects calcareous tube growth in adults and reared offspring of serpulid polychaetes.

The energetically costly transition from free-swimming larvae to a benthic life stage and maintenance of a calcareous structure can make calcifying marine invertebrates vulnerable to ocean acidification. The first goal of this study was to evaluate the impact of ocean acidification on calcified tube growth for two Serpulidae polychaete worms. Spirorbis sp. and Spirobranchus triqueter were collected at 11 m depth from the northwest Mediterranean Sea and maintained for 30 and 90 days at three mean pHT levels (total scale): 8.1 (ambient), 7.7 and 7.4. Moderately decreased tube elongation rates were observed in both species at pHT 7.7 while severe reductions occurred at pHT 7.4. There was visual evidence of dissolution and tubes were more fragile at lower pH but fragility was not attributed to changes in fracture toughness. Instead, it appeared to be due to the presence of larger alveoli covered in a thinner calcareous layer. The second objective of this study was to test for effects on S. triqueter offspring development. Spawning was induced, and offspring were reared in the same pH conditions that the parents experienced. Trochophore size was reduced at the lowest pH level but settlement success was similar across pH conditions. Post-settlement tube growth was most affected. At 38 days post-settlement, juvenile tubes at pHT 7.7 and 7.4 were half the size of those at pHT 8.1. The results suggest future carbonate chemistry will negatively affect the initiation and persistence of both biofouling and epiphytic polychaete tube worms.

Stepwise approach towards adoption of allergen immunotherapy for allergic rhinitis and asthma patients in daily practice in Belgium: a BelSACI-Abeforcal-EUFOREA statement.

Allergic rhinitis (AR) affects 23-30% of the European population with equal prevalence reported in Belgium. Despite guidelines on the correct use of effective treatment, up to 40% of AR patients remain uncontrolled. Allergen immunotherapy (AIT) has been shown to improve the level of control up to 84% of patients being controlled by AIT. Recently, new guidelines for AIT have been published, supporting the clinical evidence for effectiveness of various subcutaneous and sublingual products for AIT in patients who are allergic to airborne allergens. AIT in AR patients not only reduces nasal and/or ocular symptoms but also induces tolerance and has preventive potential. Adoption of AIT into daily clinical practice in Belgium and other European countries is hampered primarily by reimbursement issues of each of the single products but also by several patient- and physician-related factors. Patients need to be better informed about the effectiveness of AIT and the different routes of administration of AIT. Physicians dealing with AR patients should inform patients on tolerance-inducing effects of AIT and are in the need of a harmonized and practical guide that supports them in selecting eligible patients for AIT, in choosing evidence-based AIT products and in following treatment protocols with proven efficacy. Therefore, a stepwise and holistic approach is needed for better adoption of AIT in the real-life setting in Belgium.